There is considerable interest on the part of patients and healthcare providers in the development of low cost, long-acting, “user-friendly” protein therapeutics. Proteins are expensive to manufacture and unlike conventional small molecule drugs, are not readily absorbed by the body. Therefore, proteins must be administered by injection. Most proteins are cleared rapidly from the body, necessitating frequent, often daily, injections. This is particularly the case for small peptides, which often have half-lives on the order of minutes following injections into humans. Patients dislike injections, which leads to reduced compliance and reduced drug efficacy. The length of time an injected protein remains in the body is finite and is determined by the protein's size and whether or not the protein contains covalent modifications such as glycosylation.
Introduction of proteins into circulation exposes the proteins to numerous cell types, enzymes and routes of extravasation that contribute to their rapid clearance or catabolism. The protein may be attacked by plasma proteases or bind plasma proteins or cell surface receptors. Either receptor-mediated or less specific binding may result in the uptake of the protein via endocytotic or pinocytotic mechanisms, with the end result being degradation by lysosomal proteases. Proteins that avoid capture by these cells may pass out of the circulation via uptake by the liver, the lymphatic system or renal glomeruli (Sheffield, 2001).
Circulating concentrations of injected proteins change constantly, often by several orders of magnitude, over a 24 hour period. Rapidly changing concentrations of protein agonists can have dramatic downstream consequences, at times understimulating and at other times overstimulating target cells. Similar problems plague protein antagonists. These fluctuations can lead to decreased efficacy and increased frequency of adverse side-effects for protein therapeutics. The rapid clearance of recombinant proteins from the body significantly increases the amount of protein required per patient and dramatically increase the cost of treatment. Most protein products currently on the market require frequent injections, usually multiple times per week. This dosing regimen is painful, inconvenient for the patient, and may not provide the optimum therapeutic benefit. In the case of a chronic indication such as cancer or an immunodeficiency, treatment could last for years.
Thus, there is a strong need to develop protein delivery technologies that lower the costs of protein therapeutics to patients and healthcare providers. One solution to this problem is the development of methods to prolong the circulating half-lives of protein therapeutics in the body so that the proteins do not have to be injected frequently. This solution also satisfies the needs and desires of patients for protein therapeutics that are “user-friendly”, i.e., protein therapeutics that do not require frequent injections.
Many bioactive peptides have been described, including glucagon, glucagon-like peptide-1 (GLP-1), GLP-2, Gastric inhibitory peptide (GIP), PYY, exendin, ghrelin, gastrin, amylin, and oxyntomoldulin. These peptides typically are 10-40 amino acids in length. Methods to develop longer acting forms of these peptides are desired.
In addition, larger proteins, such as interferon-gamma (IFN-γ) are of high interest as therapeutics. IFN-γ was first recognized over 35 years ago on the basis of its anti-viral activity (Wheelock, 1965). Over the years, a great deal of information has accumulated that validates IFN-γ's role in modulating nearly all phases of immune and inflammatory processes. IFN-γ belongs to a family of proteins related both structurally and by their ability to protect cells from viral infection. The interferon family has three main members, now designated interferon-alpha (IFN-α), interferon-beta (IFN-β), and interferon-gamma (IFN-γ). The latter, which is also known as immune or type II interferon, has several properties related to immunoregulation that makes it different from the other IFNs. For example, IFN-γ has a 10-fold lower specific anti-viral activity than either IFN-α or IFN-β. On the other hand IFN-γ is 100-10,000 times more active as an immune system modulator than are the other classes of interferon, having potent phagocyte-activating effects not seen with other interferon types (Pace et al., 1985).
IFN-γ is a 20-25 kDa glycoprotein that exists as a homodimer in solution. Recombinant IFN-γ has an elimination half-life in the bloodstream after intravenous (iv) administration of 25-35 min and is essentially undetectable after 4 hours depending on the dose. The subcutaneous (sc) route generally results in a somewhat extended half-life of 5-6 hours (see review by Younes and Amsden, 2002). The recommended dosing schedule for IFN-γ is a sc injection of 50 μg/m2 three times weekly. Relevant to this invention is the fact that IFN-γ has a short elimination half-life, whether given iv or sc, and thus requires relatively frequent re-administration.
Several studies have demonstrated that continuous exposure to IFN-γ enhances the protein's potency. Researchers have investigated the benefits of continuous infusion of IFN-γ versus once daily intraperitoneal injections in Leishmania donavani-infected mice. Daily dosing of IFN-γ did induce anti-microbial resistance, but these effects were considerably enhanced by continuous administration of a comparable dose (47% reduction in liver parasite burden versus 9%) (Murray, 1990). In a similar study, mice were infected with Mycobacterium tuberculosis. Continuous delivery of IFN-γ via an external pump, prolonged survival longer than did daily intramuscular IFN-γ injections (12 vs. 4 days longer than controls) (Flynn et al., 1993). These studies suggest a superior therapeutic benefit from constant circulating levels of IFN-γ. Unfortunately, continuous infusion of IFN-γ is not practical for most patients. Therefore the need still exists for a long acting form of IFN-γ.
Covalent modification of proteins with polyethylene glycol (PEG) has proven to be a useful method to extend the circulating half-lives of proteins in the body (Abuchowski et al., 1984; Meyers et al., 1991; Keating et al., 1993). Several PEGylated proteins are approved for use in humans or are in human clinical trials (Harris et al., 2003). Covalent attachment of PEG to a protein increases the protein's effective size and reduces its rate of clearance from the body, presumably through interference with protein removal pathways, including kidney glomerular filtration, proteolytic degradation as well as active clearance via specific receptors.
Given the therapeutic value of long-acting forms of IFN-γ, as well as other therapeutic proteins and peptides, there is a continued need in the art to provide new variants of IFN-γ that have improved stability, higher potency, greater solubility, longer circulating half-lives for less frequent dosing, and reduced antigenicity as compared to the parent (native) protein. In addition, since expression of recombinant proteins containing free cysteine residues has been problematic due to reactivity of the free sulfhydryl at physiological conditions, there also remains a need in the art for improved, cost-effective methods for manufacturing (producing) such proteins that result in high yields of biologically active product.